Accomplishments: AIMS: To ask the questions :1)Which orphan receptors such as CAR (constitutive androstane receptor) and PXR(pregnane X receptor)(SXR)(Steroid-Xenobiotic binding Element) are involved in the regulation of the human CYP2Cs?" 2)How does human CAR differ from mouse CAR. 3)How are the CYP2Cs regulated and what nuclear factors are involved? Clinically used drugs and environmentals induce their own metabolism and cause interactions by increasing their metabolism. chemicals. Phenobarbital (PB) is a prototype of these drugs. It is now known that drugs and environmental chemicals can activate response elements in the promoter regions of many genes, including cytochromes P450 such as human CYP2B6. In addition, the nuclear receptor CAR and PXR can bind to these elements, and constitutively and inducibly activate transcription. Our laboratory is studying the regulation of the CYP2C subfamily of P450s in humans, which includes CYP2C8, CYP2C9, and CYP2C19. These are a clinically important group of P450s which metabolize about 20% of all drugs to which humans are exposed It has been shown that drugs metabolized by the human CYP2Cs show altered pharmacokinetic profiles when co-administered with PB or the antibiotic rifampicin. Experiments using HepG2 cells stably transfected with CAR demonstrated that CAR constitutively activates endogenous levels of CYP2C8 and CYP2C9 transcripts monitored by RT-PCR. This activation is inducible by PB-type inducers. CYP2C9 appears to be at least partly regulated by the PB responsive receptor CAR. However, human CAR (hCAR) appears to act differently than mouse CAR(mCAR). Moreover , CYP2C8 and CYP2C9 are differentially induced by different compounds such as phenobarbital and rifampicin indicating differences in control mechanisms. Transfection studies with luciferase reporter constructs containing the first 3 kb of of CYP2C9 have shown that CAR can activate transcription constitutively and inducibily . We have isolated one CAR binding element in CYP2C9 which appears to regulate constituitive expression. This response does not, however, totally account for the response to inducers indicating other regions may also contain elements. Deletional analysis of this CYP2C9 promoter region has localized a possible proximal region of CYP2C9 which is involved in CAR mediated activation. We have also identified a distal element of the CYP2C9 promotor that binds to hCAR>hPXR>mCAR in electrophoretic mobility shift assays. Transfection studies with this element driving a thymidine kinase promoter show that hCAR>hPXR>mCAR constitutively activates regulation of this element in HepG-2 cells.